In addition to discovering soluble small molecule for hESC/iPSC maintenance and directed differentiation, combinatorial chemistry can also serve as an excellent tool for the identification of extracellular matrix (ECM) mimics that can further facilitate the maintenance or specific- differentiation of hESC/iPSCs. The one-bead-one-compound (OBOC) combinatorial chemistry invented by Dr. Lam is particularly suited for the discovery of ECM mimics or other functional ligands against unique cell surface receptors of stem cells. We have recently developed a one- bead-two-compound (OB2C) library method by incorporating a known cell adhesion peptide (e.g. an integrin binding ligand) on the outer layer of every bead of the OBOC library. As a result, every bound cell will be exposed to the library compound displayed on the surface of each bead. We have generated genetically-labeled Olig2-GFP knock-in hESC/iPSC reporter lines that can be conveniently used as readouts in the proposed combinatorial library screening for directed-differentiation toward various lineages. Specific aims of this proposal are: 1. To design and synthesize several OBOC and OB2C combinatorial libraries for the discovery of cell surface acting molecules that can induce directed-differentiation of hESCs or iPSCs. 2. To use live Olig2-GFP knock-in reporter hESC/iPSC lines in conjunction with immunocytochemical methods to screen various OB2C libraries (from aim 1) for directed-differentiation into oligodendroglial progenitor cells. 3. To fully characterize effects of the ligands and molecules identified in aim 2 on hESC/iPSC growth and differentiation using a range of biological and biochemical approaches. To optimize and use these ligands in a novel chemically-defined self- assemble PVA-based hydrogel as artificial ECM to support stem cell growth and directed- differentiation into oligodendroglial progenitor cells [OPCs]. Impact The proposed research will enable us to discover synthetic molecules that bind to unique receptors on the surface of hESC/iPSCs. Some of these molecules may support the growth of hESC/iPSCs while maintaining its stem-ness. Others may induce specific cell signaling and oligodendrocyte differentiation. Our long term goal is to direct-differentiate oligodendrocyte precursor cells further for the treatment of neurological diseases.